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Nematode community populations in the rhizosphere of cultivated olive differs according to the plant genotype
Soil Biology & Biochemistry 45 (2012) 168e171
Contents lists available at SciVerse ScienceDirect
Soil Biology & Biochemistry journal homepage: www.elsevier.com/locate/soilbio
Short communication
Nematode community populations in the rhizosphere of cultivated olive differs according to the plant genotype Juan E. Palomares-Rius a, Pablo Castillo a, Miguel Montes-Borrego a, Henry Müller b, Blanca B. Landa a, * a b
Department of Crop Protection, Institute for Sustainable Agriculture (IAS-CSIC), Alameda del Obispo s/n, APDO. 4084, 14004 Cordoba, Spain Graz University of Technology, Institute of Environmental Biotechnology, Petersgasse 12, 8010 Graz, Austria
a r t i c l e i n f o
a b s t r a c t
Article history: Received 18 August 2011 Received in revised form 8 November 2011 Accepted 9 November 2011 Available online 23 November 2011
Classical and molecular methods were used to study the nematode communities associated with rhizosphere soil and roots of a collection of 16 olive cultivars from a world olive germplasm bank in Mengibar (Jaen province, southern Spain). Classical nematological analysis, including soil nematode extraction, species counting and morphological identification showed that 24 taxa belonging to 9 genera (including Aphelenchoides, Criconemoides, Ditylenchus, Filenchus, Helicotylenchus, Merlinius, Paratylenchus, Tylenchus, and Xiphinema) and 8 families (including Anguinidae, Aphelenchidae, Belonolaimidae, Criconematidae, Hoplolaimidae, Longidoridae, Tylenchidae and Tylenchulidae) of plant-parasitic nematodes were present, with one species (Helicotylenchus digonicus) being prevalent in all samples. The low values of the plant-parasitic nematode index (PPI) indicated a high disturbance of the field soil probably due to application of herbicides and fertilizers. Cluster analysis of population densities of the various nematode species, nematode trophic groups, and ecological indices grouped most olive cultivars into three main clusters indicating that olive genotypes differ in the nematode communities in their rhizosphere soil. The use of T-RFLP analysis discriminated to a higher extent the nematode communities present in the rhizosphere soil from the different olive cultivars as compared to the morphological-based analysis. This study provides the first evidence of an effect of the olive genotype on nematode community composition by combining classical morphological and molecular approaches. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: Helicotylenchus digonicus Nematode identification Olea europaea L. subsp. europaea var. europaea T-RFLPs 18S
For millennia, cultivated olive (Olea europaea L. subsp. europaea var. europaea) has been the main oleaginous crop of the Mediterranean Basin. Among cultivated trees, olive is one of the species of larger longevity and richest in genetic biodiversity (Rallo et al., 2000) constituting a complex of wild forms, weedy types and cultivated varieties (Aranda et al., 2011). Thus, olive cultivars show a broad range of genetic variability for a large number of agronomic traits, including oil content, vigour, fruit size, yields, and adaptability to biotic or abiotic stresses which justify the selection of the specific varieties in each region. Globally, nematodes have been recognized as good bioindicators of the soil environment (Chen et al., 2010; Ritz and Trudgill, 1999) but at the same time plant-parasitic nematodes are one of the main biotic stresses in olive (Castillo et al., 2010). Although it is known that the interaction of plant-parasitic nematodes with free-living nematodes could play an important role in their control, it is not known how olive genotype may influence the balance between plant-parasitic and free-living nematodes. Thus, whereas several
* Corresponding author. Tel.: þ34 957 4992 79; fax: þ34 957 499252. E-mail address: [email protected] (B.B. Landa). 0038-0717/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.soilbio.2011.11.009
studies have demonstrated the selective influence of specific plant genotypes, cultivars, and even ecotypes on the composition of microbial communities in the rhizosphere and endosphere (Aranda et al., 2011; Berg et al., 2005; De la Fuente et al., 2006; Nelson et al., 2011) little is known for nematode communities (Griffiths et al., 2007). The aim of this work was to study the nematode communities associated with rhizospheric soil and roots of a collection of 16 olive cultivars from a world olive germplasm bank from nine countries including very different genotypes (Fig. 1). The field was established 20 years ago at the ‘Venta del Llano’ Research Station (IFAPA, Andalusia Regional Government) in Mengibar (Jaén province, southern Spain) using olive planting stocks of the same age that were randomly distributed throughout the field. During this period all trees have received same agricultural practices, the soil is under non tillage with herbicides being applied for weed management and drip irrigation. Therefore, studying this field site will allow enlightening the specificity and effect of the cultivar genotype on nematode populations avoiding the influence of other factors associated to climatic, edaphic and agronomical conditions. Soil samples were collected in May 2009 with a shovel from the upper 30 cm of soil from four different points around each individual tree (approximately 50 cm away from the trunk). Only young
Fig. 1. Cluster analysis using the Ward algorithm of similarity among 16 olive cultivars from different geographical origin calculated using the squared Euclidean distance coefficient with Bionumerics 6.6 software (Applied Maths NV, Sint-Martens-Latem, Belgium). The intensity of grey color correlates to higher values or higher frequency. Cophenetic correlation values are indicated in each node. Groups (I, II and III) were differentiated at the cluster cutoff value indicated in each tree (*). (A) Data analyzed correspond to population number of nematode taxa identified in rhizosphere samples and of Helycotylenchus digonicus on roots. Average total number of nematodes (TNN) extracted in each cultivar is also indicated. (B) Data analyzed correspond to numbers and percentage of individual nematode trophic groups and average ecological indices including Modified Maturity index (MMI), Maturity index (MI), and Plant-Parasitic index (PPI) estimated for rhizosphere soil samples. (C) Data analyzed correspond to relative abundance of each terminal restriction fragment (TRFs) (only standardized TRFs representing at least 1% of total and present in more than 20% of samples were included) obtained from T-RFLP analysis of rhizosphere soil nematode population fingerprints generated by TaqI enzyme. Three to four independent PCR amplifications were combined before analysis for each cultivar. Three to four trees per cultivar were sampled. Richness R (total number of TRFs identified) is also indicated for each cultivar.
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J.E. Palomares-Rius et al. / Soil Biology & Biochemistry 45 (2012) 168e171
and active root samples were taken from each sampled point. Samples of soil and roots were thoroughly mixed to obtain a single representative sample per tree before extraction. Three to four trees per cultivar were sampled serving each tree as a replicate. Nematodes from the soil were extracted from a 500-cm3 subsample using the magnesium sulphate centrifugal-flotation method (Coolen, 1979). Additionally, 5 g of roots from each tree were extracted using a food blender in a previous extraction step and sieving the resulting mixture through 2 mm sieve and 5 mm mesh. Then, the procedure was the same as described for soil samples. The extracted nematodes were identified and counted as described in Castillo et al. (2010) and the solutions were left to settle during two hours at 4 C. Plant debris and other residuals were hand picked and removed using forceps, the suspension of nematodes was centrifuged at 15,000 g for 10 min, and the supernatant removed to leave the nematode pellet in approximately 50 ml water and stored frozen at 20 C. A total of 13 nematode families were detected from soil samples. Ten of these families were identified at genera or species level resulting in 15 different genus present in soil samples (Table 1). Results showed that 24 taxa belonging to 9 genera (including Aphelenchoides, Criconemoides, Ditylenchus, Filenchus, Helicotylenchus, Table 1 Frequency and population densities of nematodes in the rhizospheric soil of a collection of 16 olive cultivars in a world olive germplasm bank. Genus (species)a Soil samples Acrobeles Aphelenchoides Aphelenchus (A. avenae) Cephalobus Criconemoides xenoplax Ditylenchus Eucephalobus Filenchus (Filenchus sp., F. thornei) Helicotylenchus (H. digonicus) Merlinius (M. brevidens) Mesodorylaimus sp. Paratylenchus (P. ciccaronei, P. microdorus) Plectus Tylenchus (Tylenchus sp., T. davainei) Xiphinema (X. pachtaicum. X. nuragicum) Dorylaimidae* Plectidae* Rhabditidae* Root samples Helicotylenchus (H. digonicus) Paratylenchus (P. microdorus) Pratylenchus (P. neglectus) a
Trophic groupb
C-Pc
Frequency (%)d
Mean SD (range)e
BF FF, PP FF, PP BF PP FF, PP BF
2 2 3 2 3 2 2
8.7 4.4 37.0 2.2 13.0 6.5 10.9
FF, PP
2
82.6
PP
3
97.8
PP OM PP
2 4 3
15.2 2.2 34.9
20 11.1 (6e33) 31.5 13.4 (22e41) 31.5 36.1 (3e148) 12 9.7 13.5 (3e37) 5.7 5.5 (2e12) 146.4 118.6 (22e312) 126.2 143.2 (7e600) 5002.9 3920.7 (138e17,900) 22.6 21.0 (4e56) 21 98.5 113.9 (5e410)
BF FF, PP
2 2
2.2 71.7
79 123.7 197.4 (3e970)
PP
5
82.6
73.8 132.7 (3e600)
OM
4
97.8
BF BF
2 1
2.2 87.0
143.9 171.7 (7e1000) 3 384.6 356.4 (17e1400)
PP
3
91.3
12.8 12.2 (1e56)
PP
3
19.6
4 2.1 (1e8)
PP
3
4.4
7 1.4 (6e8)
(*) Nematodes could not be identified to the genus level. Abbreviations: BF: bacterial feeder; FF: Fungal feeder; PP: Plant-parasitic; OM: Omnivorous. c C-P: Colonizer-persister value for nematode genera (Bongers, 1990). d Determined as the percentage of samples from all replicates of the 16 olive cultivars showing presence of the nematode taxa. e Number of nematodes per 500 cc of soil. b
Merlinius, Paratylenchus, Tylenchus, and Xiphinema) and 8 families (including Anguinidae, Aphelenchidae, Belonolaimidae, Criconematidae, Hoplolaimidae, Longidoridae, Tylenchidae and Tylenchulidae) of plant-parasitic nematodes were present, with one species (Helicotylenchus digonicus) being prevalent in all samples (Table 1). On the other hand, only three species of plant parasitic nematodes (H. digonicus, Paratylenchus microdorus and Pratylenchus neglectus) were identified in roots samples. These three nematode species have been reported around and/or on olive roots and are recognized as pathogenic to olive but also may be pathogens of other vegetable and fruit crops (Vovlas et al., 2011). Helicotylenchus digonicus was predominant in the majority of samples studied (91.3%) and all olive cultivars with the exception of cv. Trylia (Table 1, Fig. 1A). Helicotylenchus digonicus is a semi-endoparasitic nematode that has been observed associated with necrosis in olive roots, and is considered capable of affecting olive tree growth under particular growing conditions (Inserra et al.,1979). Its ubiquity in the sampled olive roots may be due to the softness of the tissues of young roots and easiness of penetration, as it has been showed in other spiral species (Inserra et al., 1979). On the other hand, P. microdorus was present in less than 20% of samples and only in roots of cvs. Arbequina, Frantoio, Koroneiki, Oblonga, Ocal, and Picual, and P. neglectus was only present in a few samples (4.4%) of cvs. Koroneiki and Uslu. Olive cvs. Aglandau, Arbequina, Chétoni and Frantoio showed H. digonicus levels in their roots significantly higher (P < 0.05) than those found in cvs. Picholine, Picual and Trylia (Fig. 1A). Cluster analysis of nematode populations of all species identified in the rhizosphere soil and of H. digonicus on roots of the 16 olive cultivars grouped them into three main clusters (distance cuttoff value of 34.2%) with no clear relationship to their geographical area of cultivation (Fig. 1A). ‘Picual’, ‘Trylia’ and ‘Hojiblanca’ clearly grouped independently in cluster III differentiated by their highest levels of Dorilaimidae, Filenchus, Paratylenchus and Rabditidae. The remaining cultivars grouped in two clusters, with cluster II showing in general intermediate or higher levels of Dorilaimidae, Filenchus, and Rabditidae as compared to cultivars included in cluster III and I, respectively. It is well documented that olive trees serve as hosts to a large number of plant parasitic nematodes, of which root-knot nematodes (Meloidogyne spp.), root-lesion nematodes (Pratylenchus spp.), spiral nematodes (Helicotylenchus spp.), and Criconemoides xenoplax are widely distributed (Castillo et al., 2010; Lamberti and Vovlas, 1993; Nico et al., 2002). Those nematode species are endoparasitic forms, and many are recognized as pathogenic to olive. Conversely, limited distribution for the citrus (Tylenchulus semipenetrans) nematode has been reported in olive (Castillo et al., 2010; McKenry, 1994). Other plant-parasitic nematodes that have been identified infesting olive orchard soils in the Mediterranean Basin were also identified in this study including genera such as Paratylenchus spp., and Xiphinema spp. (Hashim 1982; Lamberti and Vovlas, 1993; Nico et al., 2002). Different nematode trophic groups [plant-parasitic (PP), fungal feeders/plant-parasitic (FF/PP), bacterial feeders (BF) and omnivorous (O)] were detected in the olive cultivars, but, strictly fungal feeders and predators were not found (Table 1; Fig. 1B). Ecological indices including Modified Maturity index (MMI), Maturity index (MI), Plant-Parasitic index (PPI) indicating the successional stage of communities, were calculated according to Bongers (1990) (Fig. 1B). Cluster analysis of the ecological indices grouped the olive cultivars within the same three main clusters than those showed in Fig. 1A, with only three exceptions (Fig. 1B). Values of the ecological indices indicated a high disturbance in the soil and an important influence of plant-parasitic nematodes in the field. High levels of soil disturbance more commonly refers to physical or chemical impacts on soil (Bongers, 1990). Agricultural practices including fertilization
J.E. Palomares-Rius et al. / Soil Biology & Biochemistry 45 (2012) 168e171
and weed control through herbicides could increase primary, root production and the levels of plant-parasitic nematodes in the studied field plot. In addition to morphological studies, DNA from nematode pellets was extracted using the PowerSoilÔ DNA Isolation Kit (MO BIO Laboratories, Inc., Carlsbad, USA) and the FastPrepÒ-24 (MP Biomedicals, Inc., Illkirch, France) instrument, using 3 cycles of 45 s burst at maximum speed (6,5X). PCR amplification of partial 18S rDNA from nematode communities were performed using the specific primer pair NEMF1/S3 following conditions described by Waite et al. (2003). Primer NEMF1 was 50 end-labeled with the fluorescent dye FAM. Terminal restriction fragment length polymorphism (T-RFLP) was performed for all samples using 5 ml of PCR products and TaqI restriction enzyme (Fast DigestÒ, Fermentas, Germany) in a final volume of 10 ml. Selection of restriction enzyme was based in preliminary ‘in-silico’ digested products from sequences of plant-parasitic nematodes (data not shown) and other references (Edel-Hermann et al., 2008). Terminal restriction fragments (TRF) were loaded and separated on a 3130XL genetic analyzer (Applied Biosystems, California, USA) at the SCAIUniversity of Córdoba sequencing facilities. Size of fragments were determined using a ROX500 size standard, and matrices containing incidence as well as peak area data of individual TRFs were generated for all samples with GeneMapper software (Applied Biosystems). TRFs profiles were selected and standardized based on methods described previously by Aranda et al. (2011). Mean Richness values ranged from 7 to 46 depending of the olive sample with an average of 23 10 (mean SD) TRFs indicated that the molecular based approach could differentiate more taxa as compared to the classical morphological analysis. However, additional work is needed to identify those taxa. Cluster analysis of standardized TRFs representing >1% of total and present in at least 20% of samples revealed three main groups (Fig. 1C) with higher variability among olive cultivars as those obtained using clasical identification methods (Fig. 1A and B). Differences between clusters obtained by using classical approaches and those revealed by TRFLP analysis must be due to non plant-parasitic nematodes or also to the constraint that identification of some species is only possible from adult specimens which usually represent only a small percentage of the overall nematode assemblage (Griffiths et al., 2002). The identification of soil nematodes often requires a high degree of taxonomic expertise, the time spent on identification of all individuals to the species level (with the corresponding costs) makes it difficult to have results over a relatively short period of time with affordability. Consequently, in general the characterization of nematode communities continues to be resolved more coarsely than at the species level (i.e. genus, family, trophic group), leaving ecological analysis potentially ambiguous or superficial (Chen et al., 2010). In our study, the use of T-RFLP analysis was demonstrated to be sufficient to discriminate and differentiated between the nematode communities present in the rhizosphere soil from different olive cultivars. Other authors have demonstrated the potential of T-RFLP procedures to differentiate nematode community structures on different habitats or as a response to organic inputs (Donn et al., 2008; Edel-Hermann et al., 2008). However, according to our knowledge this study provides the first evidence of a specific effect of the olive genotype on nematode community composition by combining classical morphological and molecular approaches. Further research should be conducted to determine how this specific selection of nematode communities may be related to olive resistance to plant parasitic nematodes.
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Acknowledgements Authors gratefully acknowledge Projects AGL2008-00344/AGR and HA2008-0014 from ‘Ministerio de Ciencia e Innovación’, Project P10-AGR-5908 from ‘Consejería de Economía, Innovación y Ciencia’ of Junta de Andalucía and FEDER financial support from the European Union. We also thank F. Gutierrez from IFAPA Venta del Llano in Mengibar and S. Aranda from sampling support.
References Aranda, S., Montes-Borrego, M., Jiménez-Díaz, R.M., Landa, B.B., 2011. Microbial communities associated with the root system of wild olives (Olea europaea L. subsp. europaea var. sylvestris) are good reservoirs of bacteria with antagonistic potential against Verticillium dahliae. Plant and Soil 343, 329e345. Berg, G., Opelt, K., Zachow, C., Lottmann, J., Götz, M., Costa, R., Smalla, K., 2005. The rhizosphere effect on bacteria antagonistic towards the pathogenic fungus Verticillium differs depending on plant species and site. FEMS Microbiology Ecology 56, 250e261. Bongers, T., 1990. The maturity index: an ecological measure of environmental disturbance based on nematode species composition. Oecologia 83, 14e19. Castillo, P., Nico, A., Navas-Cortés, J.A., Landa, B.B., Jiménez-Díaz, R.M., Vovlas, N., 2010. Plant-parasitic nematodes attacking olive trees and their management. Plant Disease 94, 148e162. Chen, X.Y., Daniell, T.J., Neilson, R., O’Flaherty, V., Griffiths, B.S., 2010. A comparison of molecular methods for monitoring soil nematodes and their use as biological indicators. European Journal of Soil Biology 46, 319e324. Coolen, W.A., 1979. Methods for extraction of Meloidogyne spp. and other nematodes from roots and soil. In: Lamberti, F., Taylor, C.E. (Eds.), Root-knot Nematodes (Meloidogyne species). Systematics, Biology and Control. Academic Press, New York, pp. 317e329. De la Fuente, L., Landa, B.B., Weller, D.M., 2006. Host crop affects rhizosphere colonization and competitiveness of 2,4-diacetyl-phloroglucinol producing Pseudomonas spp. Phytopathology 96, 751e762. Donn, S., Griffiths, B.S., Neilson, R., Daniell, T.J., 2008. DNA extraction from soil nematodes for multi-sample community studies. Applied Soil Ecology 38, 20e26. Edel-Hermann, V., Gautheron, N., Alabouvette, C., Steinberg, C., 2008. Fingerprinting methods to approach multitrophic interactions among microflora and microfauna communities in soil. Biology and Fertility of Soils 44, 975e984. Griffiths, B.S., Bengough, A.G., Neilson, R., Trudgill, D.L., 2002. The extent to which nematode communities are affected by soil factors-a pot experiment. Nematology 4, 943e952. Griffiths, B.S., Heckmann, L.H., Caul, S., Thompson, J., Scrimgeour, C., Krogh, P.H., 2007. Varietal effects of eight paired lines of transgenic Bt maize and nearisogenic non-Bt maize on soil microbial and nematode community structure. Plant Biotechnology Journal 5, 60e68. Hashim, Z., 1982. Distribution, pathogenicity and control of nematodes associated with olive. Revue de Nématologie 5, 169e181. Inserra, R.N., Vovlas, N., Golden, A.M., 1979. Helicotylenchus oleae n. sp. and H. neopaxilli n. sp. (Hoplolaimidae). Two new spiral nematodes parasitic in olive trees in Italy. Journal of Nematology 11, 56e62. Lamberti, F., Vovlas, N., 1993. Plant parasitic nematodes associated with olive. Bulletin OEPP/EPPO 23, 481e488. McKenry, M.V., 1994. Nematodes of olive. In: Ferguson, L., Sibett, G.S., Martin, G.C. (Eds.), Olive Production Manual. University of California, Oakland, pp. 97e99. Publ. 3353. Nelson, A.G., Quideau, S., Frick, B., Niziol, D., Clapperton, J., Spaner, D., 2011. Spring wheat genotypes differentially alter soil microbial communities and wheat breadmaking quality in organic and conventional systems. Canadian Journal of Plant Science 91, 485e495. Nico, A.I., Rapoport, H.F., Jiménez-Díaz, R.M., Castillo, P., 2002. Incidence and population density of plant-parasitic nematodes associated with olive planting stocks at nurseries in southern Spain. Plant Disease 86, 1075e1079. Rallo, P., Dorado, G., Martín, A., 2000. Development of simple sequence repeats (SSRs) in olive tree (Olea europaea L.). Theoretical and Applied Genetics 101, 984e989. Ritz, K., Trudgill, D.L., 1999. Utility of nematode community analysis as an integrated measure of functional state of soils: perspectives and challenges. Plant and Soil 212, 1e11. Vovlas, N., Castillo, P., Troccoli, A., 2011. Plant-parasitic nematodes of olive. In: Schena, L., Agosteo, G.E., Cacciola, S.O. (Eds.), Olive Diseases and Disorders. Transworld Research Network, Kerala, India, pp. 329e356. Waite, I.S., O’Donnell, A.G., Harrison, A., Davies, J.T., Colvan, S.R., Ekschmitt, K., Dogan, H., Wolters, V., Bongers, T., Bongers, M., Bakonyi, G., Nagy, P., Papatheodorou, E.M., Stamou, G.P., Bostrom, S., 2003. Design and evaluation of nematode 18S rDNA primers for PCR and denaturing gradient gel electrophoresis (DGGE) of soil community DNA. Soil Biology and Biochemistry 35, 1165e1173.
Contents lists available at SciVerse ScienceDirect
Soil Biology & Biochemistry journal homepage: www.elsevier.com/locate/soilbio
Short communication
Nematode community populations in the rhizosphere of cultivated olive differs according to the plant genotype Juan E. Palomares-Rius a, Pablo Castillo a, Miguel Montes-Borrego a, Henry Müller b, Blanca B. Landa a, * a b
Department of Crop Protection, Institute for Sustainable Agriculture (IAS-CSIC), Alameda del Obispo s/n, APDO. 4084, 14004 Cordoba, Spain Graz University of Technology, Institute of Environmental Biotechnology, Petersgasse 12, 8010 Graz, Austria
a r t i c l e i n f o
a b s t r a c t
Article history: Received 18 August 2011 Received in revised form 8 November 2011 Accepted 9 November 2011 Available online 23 November 2011
Classical and molecular methods were used to study the nematode communities associated with rhizosphere soil and roots of a collection of 16 olive cultivars from a world olive germplasm bank in Mengibar (Jaen province, southern Spain). Classical nematological analysis, including soil nematode extraction, species counting and morphological identification showed that 24 taxa belonging to 9 genera (including Aphelenchoides, Criconemoides, Ditylenchus, Filenchus, Helicotylenchus, Merlinius, Paratylenchus, Tylenchus, and Xiphinema) and 8 families (including Anguinidae, Aphelenchidae, Belonolaimidae, Criconematidae, Hoplolaimidae, Longidoridae, Tylenchidae and Tylenchulidae) of plant-parasitic nematodes were present, with one species (Helicotylenchus digonicus) being prevalent in all samples. The low values of the plant-parasitic nematode index (PPI) indicated a high disturbance of the field soil probably due to application of herbicides and fertilizers. Cluster analysis of population densities of the various nematode species, nematode trophic groups, and ecological indices grouped most olive cultivars into three main clusters indicating that olive genotypes differ in the nematode communities in their rhizosphere soil. The use of T-RFLP analysis discriminated to a higher extent the nematode communities present in the rhizosphere soil from the different olive cultivars as compared to the morphological-based analysis. This study provides the first evidence of an effect of the olive genotype on nematode community composition by combining classical morphological and molecular approaches. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: Helicotylenchus digonicus Nematode identification Olea europaea L. subsp. europaea var. europaea T-RFLPs 18S
For millennia, cultivated olive (Olea europaea L. subsp. europaea var. europaea) has been the main oleaginous crop of the Mediterranean Basin. Among cultivated trees, olive is one of the species of larger longevity and richest in genetic biodiversity (Rallo et al., 2000) constituting a complex of wild forms, weedy types and cultivated varieties (Aranda et al., 2011). Thus, olive cultivars show a broad range of genetic variability for a large number of agronomic traits, including oil content, vigour, fruit size, yields, and adaptability to biotic or abiotic stresses which justify the selection of the specific varieties in each region. Globally, nematodes have been recognized as good bioindicators of the soil environment (Chen et al., 2010; Ritz and Trudgill, 1999) but at the same time plant-parasitic nematodes are one of the main biotic stresses in olive (Castillo et al., 2010). Although it is known that the interaction of plant-parasitic nematodes with free-living nematodes could play an important role in their control, it is not known how olive genotype may influence the balance between plant-parasitic and free-living nematodes. Thus, whereas several
* Corresponding author. Tel.: þ34 957 4992 79; fax: þ34 957 499252. E-mail address: [email protected] (B.B. Landa). 0038-0717/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.soilbio.2011.11.009
studies have demonstrated the selective influence of specific plant genotypes, cultivars, and even ecotypes on the composition of microbial communities in the rhizosphere and endosphere (Aranda et al., 2011; Berg et al., 2005; De la Fuente et al., 2006; Nelson et al., 2011) little is known for nematode communities (Griffiths et al., 2007). The aim of this work was to study the nematode communities associated with rhizospheric soil and roots of a collection of 16 olive cultivars from a world olive germplasm bank from nine countries including very different genotypes (Fig. 1). The field was established 20 years ago at the ‘Venta del Llano’ Research Station (IFAPA, Andalusia Regional Government) in Mengibar (Jaén province, southern Spain) using olive planting stocks of the same age that were randomly distributed throughout the field. During this period all trees have received same agricultural practices, the soil is under non tillage with herbicides being applied for weed management and drip irrigation. Therefore, studying this field site will allow enlightening the specificity and effect of the cultivar genotype on nematode populations avoiding the influence of other factors associated to climatic, edaphic and agronomical conditions. Soil samples were collected in May 2009 with a shovel from the upper 30 cm of soil from four different points around each individual tree (approximately 50 cm away from the trunk). Only young
Fig. 1. Cluster analysis using the Ward algorithm of similarity among 16 olive cultivars from different geographical origin calculated using the squared Euclidean distance coefficient with Bionumerics 6.6 software (Applied Maths NV, Sint-Martens-Latem, Belgium). The intensity of grey color correlates to higher values or higher frequency. Cophenetic correlation values are indicated in each node. Groups (I, II and III) were differentiated at the cluster cutoff value indicated in each tree (*). (A) Data analyzed correspond to population number of nematode taxa identified in rhizosphere samples and of Helycotylenchus digonicus on roots. Average total number of nematodes (TNN) extracted in each cultivar is also indicated. (B) Data analyzed correspond to numbers and percentage of individual nematode trophic groups and average ecological indices including Modified Maturity index (MMI), Maturity index (MI), and Plant-Parasitic index (PPI) estimated for rhizosphere soil samples. (C) Data analyzed correspond to relative abundance of each terminal restriction fragment (TRFs) (only standardized TRFs representing at least 1% of total and present in more than 20% of samples were included) obtained from T-RFLP analysis of rhizosphere soil nematode population fingerprints generated by TaqI enzyme. Three to four independent PCR amplifications were combined before analysis for each cultivar. Three to four trees per cultivar were sampled. Richness R (total number of TRFs identified) is also indicated for each cultivar.
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and active root samples were taken from each sampled point. Samples of soil and roots were thoroughly mixed to obtain a single representative sample per tree before extraction. Three to four trees per cultivar were sampled serving each tree as a replicate. Nematodes from the soil were extracted from a 500-cm3 subsample using the magnesium sulphate centrifugal-flotation method (Coolen, 1979). Additionally, 5 g of roots from each tree were extracted using a food blender in a previous extraction step and sieving the resulting mixture through 2 mm sieve and 5 mm mesh. Then, the procedure was the same as described for soil samples. The extracted nematodes were identified and counted as described in Castillo et al. (2010) and the solutions were left to settle during two hours at 4 C. Plant debris and other residuals were hand picked and removed using forceps, the suspension of nematodes was centrifuged at 15,000 g for 10 min, and the supernatant removed to leave the nematode pellet in approximately 50 ml water and stored frozen at 20 C. A total of 13 nematode families were detected from soil samples. Ten of these families were identified at genera or species level resulting in 15 different genus present in soil samples (Table 1). Results showed that 24 taxa belonging to 9 genera (including Aphelenchoides, Criconemoides, Ditylenchus, Filenchus, Helicotylenchus, Table 1 Frequency and population densities of nematodes in the rhizospheric soil of a collection of 16 olive cultivars in a world olive germplasm bank. Genus (species)a Soil samples Acrobeles Aphelenchoides Aphelenchus (A. avenae) Cephalobus Criconemoides xenoplax Ditylenchus Eucephalobus Filenchus (Filenchus sp., F. thornei) Helicotylenchus (H. digonicus) Merlinius (M. brevidens) Mesodorylaimus sp. Paratylenchus (P. ciccaronei, P. microdorus) Plectus Tylenchus (Tylenchus sp., T. davainei) Xiphinema (X. pachtaicum. X. nuragicum) Dorylaimidae* Plectidae* Rhabditidae* Root samples Helicotylenchus (H. digonicus) Paratylenchus (P. microdorus) Pratylenchus (P. neglectus) a
Trophic groupb
C-Pc
Frequency (%)d
Mean SD (range)e
BF FF, PP FF, PP BF PP FF, PP BF
2 2 3 2 3 2 2
8.7 4.4 37.0 2.2 13.0 6.5 10.9
FF, PP
2
82.6
PP
3
97.8
PP OM PP
2 4 3
15.2 2.2 34.9
20 11.1 (6e33) 31.5 13.4 (22e41) 31.5 36.1 (3e148) 12 9.7 13.5 (3e37) 5.7 5.5 (2e12) 146.4 118.6 (22e312) 126.2 143.2 (7e600) 5002.9 3920.7 (138e17,900) 22.6 21.0 (4e56) 21 98.5 113.9 (5e410)
BF FF, PP
2 2
2.2 71.7
79 123.7 197.4 (3e970)
PP
5
82.6
73.8 132.7 (3e600)
OM
4
97.8
BF BF
2 1
2.2 87.0
143.9 171.7 (7e1000) 3 384.6 356.4 (17e1400)
PP
3
91.3
12.8 12.2 (1e56)
PP
3
19.6
4 2.1 (1e8)
PP
3
4.4
7 1.4 (6e8)
(*) Nematodes could not be identified to the genus level. Abbreviations: BF: bacterial feeder; FF: Fungal feeder; PP: Plant-parasitic; OM: Omnivorous. c C-P: Colonizer-persister value for nematode genera (Bongers, 1990). d Determined as the percentage of samples from all replicates of the 16 olive cultivars showing presence of the nematode taxa. e Number of nematodes per 500 cc of soil. b
Merlinius, Paratylenchus, Tylenchus, and Xiphinema) and 8 families (including Anguinidae, Aphelenchidae, Belonolaimidae, Criconematidae, Hoplolaimidae, Longidoridae, Tylenchidae and Tylenchulidae) of plant-parasitic nematodes were present, with one species (Helicotylenchus digonicus) being prevalent in all samples (Table 1). On the other hand, only three species of plant parasitic nematodes (H. digonicus, Paratylenchus microdorus and Pratylenchus neglectus) were identified in roots samples. These three nematode species have been reported around and/or on olive roots and are recognized as pathogenic to olive but also may be pathogens of other vegetable and fruit crops (Vovlas et al., 2011). Helicotylenchus digonicus was predominant in the majority of samples studied (91.3%) and all olive cultivars with the exception of cv. Trylia (Table 1, Fig. 1A). Helicotylenchus digonicus is a semi-endoparasitic nematode that has been observed associated with necrosis in olive roots, and is considered capable of affecting olive tree growth under particular growing conditions (Inserra et al.,1979). Its ubiquity in the sampled olive roots may be due to the softness of the tissues of young roots and easiness of penetration, as it has been showed in other spiral species (Inserra et al., 1979). On the other hand, P. microdorus was present in less than 20% of samples and only in roots of cvs. Arbequina, Frantoio, Koroneiki, Oblonga, Ocal, and Picual, and P. neglectus was only present in a few samples (4.4%) of cvs. Koroneiki and Uslu. Olive cvs. Aglandau, Arbequina, Chétoni and Frantoio showed H. digonicus levels in their roots significantly higher (P < 0.05) than those found in cvs. Picholine, Picual and Trylia (Fig. 1A). Cluster analysis of nematode populations of all species identified in the rhizosphere soil and of H. digonicus on roots of the 16 olive cultivars grouped them into three main clusters (distance cuttoff value of 34.2%) with no clear relationship to their geographical area of cultivation (Fig. 1A). ‘Picual’, ‘Trylia’ and ‘Hojiblanca’ clearly grouped independently in cluster III differentiated by their highest levels of Dorilaimidae, Filenchus, Paratylenchus and Rabditidae. The remaining cultivars grouped in two clusters, with cluster II showing in general intermediate or higher levels of Dorilaimidae, Filenchus, and Rabditidae as compared to cultivars included in cluster III and I, respectively. It is well documented that olive trees serve as hosts to a large number of plant parasitic nematodes, of which root-knot nematodes (Meloidogyne spp.), root-lesion nematodes (Pratylenchus spp.), spiral nematodes (Helicotylenchus spp.), and Criconemoides xenoplax are widely distributed (Castillo et al., 2010; Lamberti and Vovlas, 1993; Nico et al., 2002). Those nematode species are endoparasitic forms, and many are recognized as pathogenic to olive. Conversely, limited distribution for the citrus (Tylenchulus semipenetrans) nematode has been reported in olive (Castillo et al., 2010; McKenry, 1994). Other plant-parasitic nematodes that have been identified infesting olive orchard soils in the Mediterranean Basin were also identified in this study including genera such as Paratylenchus spp., and Xiphinema spp. (Hashim 1982; Lamberti and Vovlas, 1993; Nico et al., 2002). Different nematode trophic groups [plant-parasitic (PP), fungal feeders/plant-parasitic (FF/PP), bacterial feeders (BF) and omnivorous (O)] were detected in the olive cultivars, but, strictly fungal feeders and predators were not found (Table 1; Fig. 1B). Ecological indices including Modified Maturity index (MMI), Maturity index (MI), Plant-Parasitic index (PPI) indicating the successional stage of communities, were calculated according to Bongers (1990) (Fig. 1B). Cluster analysis of the ecological indices grouped the olive cultivars within the same three main clusters than those showed in Fig. 1A, with only three exceptions (Fig. 1B). Values of the ecological indices indicated a high disturbance in the soil and an important influence of plant-parasitic nematodes in the field. High levels of soil disturbance more commonly refers to physical or chemical impacts on soil (Bongers, 1990). Agricultural practices including fertilization
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and weed control through herbicides could increase primary, root production and the levels of plant-parasitic nematodes in the studied field plot. In addition to morphological studies, DNA from nematode pellets was extracted using the PowerSoilÔ DNA Isolation Kit (MO BIO Laboratories, Inc., Carlsbad, USA) and the FastPrepÒ-24 (MP Biomedicals, Inc., Illkirch, France) instrument, using 3 cycles of 45 s burst at maximum speed (6,5X). PCR amplification of partial 18S rDNA from nematode communities were performed using the specific primer pair NEMF1/S3 following conditions described by Waite et al. (2003). Primer NEMF1 was 50 end-labeled with the fluorescent dye FAM. Terminal restriction fragment length polymorphism (T-RFLP) was performed for all samples using 5 ml of PCR products and TaqI restriction enzyme (Fast DigestÒ, Fermentas, Germany) in a final volume of 10 ml. Selection of restriction enzyme was based in preliminary ‘in-silico’ digested products from sequences of plant-parasitic nematodes (data not shown) and other references (Edel-Hermann et al., 2008). Terminal restriction fragments (TRF) were loaded and separated on a 3130XL genetic analyzer (Applied Biosystems, California, USA) at the SCAIUniversity of Córdoba sequencing facilities. Size of fragments were determined using a ROX500 size standard, and matrices containing incidence as well as peak area data of individual TRFs were generated for all samples with GeneMapper software (Applied Biosystems). TRFs profiles were selected and standardized based on methods described previously by Aranda et al. (2011). Mean Richness values ranged from 7 to 46 depending of the olive sample with an average of 23 10 (mean SD) TRFs indicated that the molecular based approach could differentiate more taxa as compared to the classical morphological analysis. However, additional work is needed to identify those taxa. Cluster analysis of standardized TRFs representing >1% of total and present in at least 20% of samples revealed three main groups (Fig. 1C) with higher variability among olive cultivars as those obtained using clasical identification methods (Fig. 1A and B). Differences between clusters obtained by using classical approaches and those revealed by TRFLP analysis must be due to non plant-parasitic nematodes or also to the constraint that identification of some species is only possible from adult specimens which usually represent only a small percentage of the overall nematode assemblage (Griffiths et al., 2002). The identification of soil nematodes often requires a high degree of taxonomic expertise, the time spent on identification of all individuals to the species level (with the corresponding costs) makes it difficult to have results over a relatively short period of time with affordability. Consequently, in general the characterization of nematode communities continues to be resolved more coarsely than at the species level (i.e. genus, family, trophic group), leaving ecological analysis potentially ambiguous or superficial (Chen et al., 2010). In our study, the use of T-RFLP analysis was demonstrated to be sufficient to discriminate and differentiated between the nematode communities present in the rhizosphere soil from different olive cultivars. Other authors have demonstrated the potential of T-RFLP procedures to differentiate nematode community structures on different habitats or as a response to organic inputs (Donn et al., 2008; Edel-Hermann et al., 2008). However, according to our knowledge this study provides the first evidence of a specific effect of the olive genotype on nematode community composition by combining classical morphological and molecular approaches. Further research should be conducted to determine how this specific selection of nematode communities may be related to olive resistance to plant parasitic nematodes.
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Acknowledgements Authors gratefully acknowledge Projects AGL2008-00344/AGR and HA2008-0014 from ‘Ministerio de Ciencia e Innovación’, Project P10-AGR-5908 from ‘Consejería de Economía, Innovación y Ciencia’ of Junta de Andalucía and FEDER financial support from the European Union. We also thank F. Gutierrez from IFAPA Venta del Llano in Mengibar and S. Aranda from sampling support.
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